Physiology&Behavior,Vol. 50, pp. 47-52. ©Pergamon Press pie. 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

Heart Rate Response of the Rat Fetus and Neonate to a Chemosensory Stimulus W I L L I A M P. S M O T H E R M A N , 1 S C O q ' T R. R O B I N S O N

Center for Developmental Psychobiology Department of Psychology, SUNY-Binghamton, Binghamton, NY 13902-6000 A P R I L E. R O N C A , J E F F R E Y R. A L B E R T S

Department of Psychology, Indiana University, Bloomington, IN 47405 AND P E T E R G. H E P P E R

School of Psychology, The Queen's University of Belfast, Belfast, Northern Ireland, UK BT7 INN R e c e i v e d 5 N o v e m b e r 1990

SMOTHERMAN, W. P., S. R. ROBINSON, A. E. RONCA, J. R. ALBERTS AND P. G. HEPPER. Heart rate response of the rat fetus and neonate to a chemosensory stimulus. PHYSIOL BEHAV 50(1) 47-52, 1991.--Resting heart rate (HR) and HR responses of fetal and neonatal rats are described before and after intraoral infusion of isotonic saline or lemon solution. Stable measurements of resting HR were obtained for fetuses over the last three days of gestation (El9, E20, E21) and pups on the day of birth (P0) and four subsequent postnatal ages (P1, P3, P5, P7). Resting HR decreased significantly on P0 relative to the three prenatal ages and exhibited a linear increase thereafter. Variability in resting HR was pronounced on E21, decreased sharply after birth, and gradually increased through P7. Developmental changes in the HR response of fetuses and pups were evident following infusion of lemon. Fetal HR responses to lemon were characterized by bradycardia, which increased in magnitude through P1, diminished after P1, and eventually changed to tachycardia by P7. Both resting HR and HR responses to chemosensory stimulation point to the immediate perinatal period as a time of quantitative and qualitative change during sensory development. Perinatal period

Chemosensory stimulation

Heart rate

Fetal heart rate

Sensory development

stimuli (10, 21, 39) are complementing a growing neurobiological literature concerned with the early development and functional activity of chemosensory systems (7,24). Providing perinatal subjects with chemosensory stimuli has proven effective in unmasking early behavioral organization or competence. The developmental origin of chemosensory-evoked behavioral responses recently has been traced to the prenatal period. By day 19 of a 21.5 day gestation, rat fetuses exhibit a general increase in motor activity in response to a wide range of chemical stimuli, including milk and lemon (34). Specific behavioral responses to milk and lemon are expressed 24 h later (33). Intraoral infusion of milk to 20-day-old fetuses elicits a stereotypic stretch response that closely resembles the behavior expressed by pups receiving milk at the nipple (9,22). Exposure to

NEWBORN altricial rodents, such as rat pups, appear helpless but in fact possess a sophisticated behavioral repertoire that enables them to deal with the changing demands of their postnatal environment (4,20). The ingestive behavior associated with suckling is a clear example of early behavioral sophistication. Newborn rats can maintain an orientation relative to the mother, locate and attach to a nipple, and suckle to elicit milk letdown (6). Aversive responses to appropriate stimulation, while not ordinarily expressed in the nest environment, are also evident as early as one day after birth. Certain components of adult aversion reactions, such as gaping, forelimb flailing, and suppressed intake can be evoked from one-day-old rat pups exposed to strong quinine or acid solutions (17). Behavioral findings that young mammals are highly responsive to olfactory or gustatory

1Requests for reprints should be addressed to William P. Smotherman, Center for Developmental Psychobiology, Department of Psychology, P.O. Box 6000, SUNY-Binghamton, Binghamton, NY 13902--6000.

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48

PERINATAL CARDIAC RESPONSES

a lemon solution at this age elicits the equally distinctive behavior of facial wiping, which appears to correspond to components of adult aversion and grooming sequences (11,12). This behavioral evidence demonstrates that the fetus can detect, distinguish, and selectively respond to different classes of chemical stimuli in utero. Functional expression of chemosensory-evoked behavior by fetuses and neonates implies continuity over the perinatal period. However, reliance on motor performances to measure sensory responsiveness during the perinatal period is complicated by changes in the physical environment associated with birth. For instance, the tendency for newborn rats to express the facial wiping response after lemon infusion is contingent upon testing in an environmental context that permits forelimb-facial contact (e.g., immersion in a buoyant fluid) (36). Asking specific questions about behavioral continuity is complicated because experimental procedures and behavioral measures appropriate for one period of development may be inappropriate at other ages (3,40). Objective resolution of issues of continuity necessitates that methods be adopted that can be applied at disparate points in development. Phasic changes in HR have figured prominently in psychophysiological research on postnatal subjects as a measure of sensory responsiveness. Cardiac responses of developing organisms can reveal early sensory competence (15, 30, 42) and reflect differential experience with stimuli (23, 31, 41). Because techniques for recording fetal cardiac reactivity can be employed across a great diversity of species (2,18), it is especially promising as a tool in the study of sensory development. Further, HR can be recorded from both pups (29) and fetuses (35). In a recent report, HR was successfully employed to measure chemosensory responsiveness in term and caesarean-derived preterm rat pups (28), corroborating the utility of this metric in studies conducted during the perinatal period. In the present study, the cardiac responsiveness of rat fetuses and rat pups to infusions of chemosensory fluids was investigated. METHOD

(El9, N=23, E20, N=25, E21, N=23, P0, N=20, P1, N = 19, P3, N = 20, P5, N = 20 and P7, N = 22). To eliminate the confounding of treatment with litter effects, no more than one animal from a given mother was tested in a particular treatment condition. A total of 116 dams provided fetal and neonatal subjects in this study.

Prenatal Preparation To record fetal HR, pregnant females were prepared for testing of fetuses on embryonic day El9, E20, or E21 according to procedures fully described by Smotherman, Richards and Robinson (32). Pregnant females were briefly anesthetized using ether and spinal anesthesia was induced by injection of 100% ethanol between the first and second lumbar vertebrae. This procedure eliminates sensation in the lower body. The female was then placed in a Plexiglas holding apparatus in a bath of buffered isotonic saline (37.5°C) immersing the lower body. The uterus was then externalized by midline laparotomy. The mother and fetuses were left for 20 minutes to recover from the ether anesthesia. Subject fetuses were delivered from the uterus and amniotic sac into the bath, but remained attached by means of the umbilical cord to the placenta, which remained within the uterus. The coloration of the umbilical cord and fetus was visually assessed during the experiment to ensure that the fetus remained fully oxygenated.

Postnatal Preparation Females and offspring were left undisturbed after birth (vaginal delivery) until the time for testing on postnatal day P0, P1, P3, P5, or P7. Experiments conducted on the day of birth (day P0) involved testing pups prior to suckling experience. Litters were separated from dams and placed in a warm humid incubator (32.5°C), where they remained for 30-60 min prior to testing. All testing occurred between 1300-1700. Postnatal testing was conducted after placement of individual subjects in a small (12 × 12 cm) plastic-floored testing arena maintained at 32.5°C.

Subjects

HR Recording

Adult female Sprague-Dawley rats (Charles-River Labs., Wilmington, MA) were maintained under conditions of constant room temperature (22°C) on a 12-h light:dark cycle (lights on at 0700). Food and water were freely available. Females were bred with Long-Evans male rats to produce animals for the experiments. Dally vaginal smears during the period of breeding were examined to date conception (the presence of sperm in a smear was designated as the start of pregnancy, E0). Breeding females were housed in groups of three in polycarbonate cages (33 x 38 × 10 cm). For pups to be tested after birth, females were rehoused individually one day prior to parturition. Animals were maintained and used in accordance with the NIH Guide for the Care and Use of Laboratory Animals (PHS Publication No. 86-23). Each fetal or neonatal subject was fitted with cardiac leads and tested at only one age. Resting HR and HR variability were measured in a total of 486 fetuses and pups. The large sample sizes tested at each age reflects the fact that collection of resting HR data has been a standard protocol in our laboratory. The sample sizes tested at each age (El9, N=40, E20, N=42, E21, N=60, P0, N=81, P1, N = 100, P3, N=100, PS, N = 3 0 and P7, N = 33) were collected from the preinfusion period of subjects in the present study as well as data from several normative studies conducted in our laboratory. HR responses to chemosensory infusion were measured in a total of 172 fetuses and pups

Fetuses and pups were fitted with paired cardiac leads fashioned by stripping the terminal insulation from #36 nickelchrome wire. The tips of the two leads were bent to a sharp angle, coated with lidocaine, and inserted under the skin ventrally (overlying the sternum) and dorsally (overlying the thoracic vertebrae). The leads were connected to a Grass model 79 polygraph, which amplified the EKG signal and provided a permanent strip record of individual heart beats (35). This strip record was divided into successive 5-s periods and the number of beats counted to calculate HR over the session. Recording commenced after a delay of 2 rain in the testing environment.

Cannulation Controlled presentation of stimuli into the mouth of individual rats was accomplished through the use of an intraoral cannula (14). The cannula consisted of a length of PE-10 polyethylene tubing (outer diameter=0.61 mm) inserted through the midline of the lower jaw with the flanged tip resting on the dorsal surface of the tongue. The position on the tongue was equivalent to the anterior placement described by Kehoe and Blass (19). The free end of the cannula was connected by way of a length of PE-50 tubing to a micrometer syringe. This system enabled precise infusion ( _ 1 ILl) of a solution to the animal without otherwise interrupting ongoing activity. Infusions were

SMOTHERMAN ET AL.

49

delivered in a 1-2-s pulse. Animals at ages El9, E20, E21 received 20 I~1 infusions, pups at ages P0, P1 and P3 received 30 }zl infusions, pups at ages P5 and P7 received 40 p,l and 50 Ixl infusions, respectively. Solutions delivered by intraoral infusion consisted of isotonic saline or lemon extract presented in an isotonic saline carrier. The lemon solution was prepared by mixing one part of pure lemon extract (Schilling brand) in three parts saline, centrifuging and removing the supernatant oil (33), Test solutions were delivered at ambient testing temperature: 37.5°C for fetuses and 32.5°(3 for pups.

400' 360

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Prenatal

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Data Analysis

E19

HR was continuously recorded for 55 seconds before infusion and 30 seconds after infusion. All fetuses and pups included in these samples were fitted with an intraoral carmula, but did not receive an infusion during the collection of resting HR data. These data were analyzed in a one-way (8 ages) between-subjects ANOVA. HR was calculated for each of the 11 5-s intervals before infusion. HR scores in successive 5-s intervals were compared to measure HR variability. Transitions between intervals in which HR changed by more than 5% were considered instances of high HR variability. To measure cardiac response to infusion, the postinfusion period was divided into six 5-s intervals to permit analysis of cardiac responsiveness to stimulation. For each subject, a series of change scores was calculated as the difference between absolute HR during the 5-s interval and the baseline HR expressed during the 5-s interval immediately preceding the infusion. Change scores were compared in a three-factor (8 ages x 2 stimuli x 6 intervals) repeated-measures ANOVA. The purpose of this analysis was to characterize the temporal pattern of HR response at different ages following saline or lemon infusion. A second analysis was employed to determine whether a significant change in HR occurred after infusion. In this analysis, the 5-s interval exhibiting the largest change in HR was defined as the Peak Interval. Data from the Peak Interval were compared in a two-factor (8 ages × 2 stimuli) ANOVA. Comparison of the peak change in HR to baseline HR permitted identification of ages at which lemon or saline infusion evoked significant change in HR. This comparison was performed by constructing separate 99% confidence intervals around preinfusion baseline HR for each age. HR change scores that lay more than one standard error above or below the confidence interval were interpreted as evidence of significant cardiac response to infusion. Correlation coefficients were calculated to determine whether the magnitude of cardiac response was influenced by the baseline HR of each subject (26).

Resting HR in the Fetal and Neonatal Rat Analysis of resting HR indicated the significant effect of age, F(7,478)---73.0, p0.10). DISCUSSION

Heart rate provides a useful metric for assessing responsiveness to sensory stimulation during the perinatal period. The techniques employed provide accurate and stable measurements of HR during both the prenatal and immediate postnatal periods.

SMOTHERMAN ET AL.

51

sot Saline 0"

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Age (days) FIG. 4. Peak change in HR of rat fetuses and pups following infusion of saline (top panel) or lemon (bottom panel). Bars represent the HR change score (mean-SE) of greatest magnitudeduring the 30 s following infusion. Data are plotted separately for prenatal (E19-E21) and postnatal (P0--PT) subjects. The zero line represents resting FIR during the 55 s prior to infusion, with the shaded area depictingthe 99% confidence limit around this resting HR at each age. Values for resting HR on P0 and P1 replicate HR measurements reported by Ronca and Alberts (28), and data for older pups are similar to values reported by Wigal, Dailey and Amsel (42). The resting HR of fetuses that retain placental-uterine attachment was markedly higher than caesarean-derived preterm pups tested at the same postconceptional age (28), but accorded well with recent HR measurements obtained from rat fetuses by ultrasonography (25) and with older data from rat fetuses adjusted for differences in body temperature (1). The increased variability in HR on E21 was noteworthy, and probably was due to uterine contractility in advance of labor. The gradual increase in postnatal HR variability (most evident on P3-P7) may have been due to differences in overall motor activity exhibited by pups. Chemosensory infusion was effective in eliciting cardiac responses from both fetal and neonatal rats. The pattern of these HR responses varied as a function of age. Bradycardia in response to lemon infusion was most pronounced at term and during the first 24 hours after birth. Although resting HR also was found to vary with age, there was no evident relationship between resting HR and the magnitude of cardiac response after infusion. This finding contrasts with the data of Ronca and AIberts (28), who reported that HR responses of caesarean-derived preterm rat pups covaried with resting HR; pups with greater resting FIR exhibited less pronounced bradycardia in response to lemon infusion. This discrepancy may reflect the impaired physiological status of preterm-delivered pups, which suggests that baseline HR may be relevant in interpreting sensory-evoked HR responses of premature subjects. Other studies reporting cardiac responses during the perinatal

period have focused on older pups [e.g., (42)], have averaged response data over a large number of stimulus presentations (8), or have tested pups after varying periods of maternal and food deprivation (16). What is striking about the present results, in light of published data, is the magnitude of HR change evoked by a single exposure to a novel chemosensory stimulus. For example, Dailey and Amsel (8) report a maximal FIR deviation from baseline of 2-3% averaged over 60 milk infusion trials in 8-day-old pups. Small changes in HR following exposure to odorants also have been reported in rats on P2-P3 (5,23). In contrast, this study found a 25% change in HR on P1 [cf. (28)]. This pronounced cardiac response was evident before birth as well, as illustrated by a 20% HR deviation from baseline in 21day-old fetuses exposed to a single lemon infusion. Pups on P0 exhibited bradycardia to both lemon and saline infusion, an event that constituted their first exposure to a fluid in the mouth after the onset of breathing. Introduction of a fluid in the upper respiratory tract is known to evoke bradycardia in newborn lambs (13). Bradycardia evoked by infusion of a lemon solution is associated with respiratory slowing in newborn rats (28). Evidence from fetuses and pups suggests that behavioral responses after infusion are evoked by chemosensory properties of the lemon solution (37). However, the HR response to saline, which is evident in newborn pups (P0) but is not expressed by fetuses of the same postconceptional age (E21), diminishes in magnitude over the first few days of postnatal life (P0-P3). This developmental change in HR response to saline may reflect the pup's increasing ability to maintain adequate ventilation while processing a fluid in the oral cavity. During the first hours after birth, the newborn must initiate air breathing and coordinate this activity with suckling. These findings suggest the hypothesis that the newborn's cardiac responsiveness to fluid stimuli is influenced by the parallel development of breathing, suckling, the interaction of these activities, and perhaps other processes involved in the transition between prenatal and postnatal life. The cardiac response to saline infusion and the suppression in resting FIR that are evident on the day of birth probably reflect the physiological adaptation of the newborn rat to an airbreathing existence. In contrast, an inflection point is apparent on P1 between two developmental trends in cardiac response to lemon infusion. Prior to P1, the bradycardia evoked by a lemon stimulus increases in magnitude with advancing age. After P1, the pattern of cardiac response to chemosensory stimulation gradually shifts to a biphasic response comprising both bradycardia and tachycardia (PS) and ultimately to a uniform tachycardia (P7), Because the inflection point in cardiac response to lemon infusion is delayed relative to the time of birth, it seems unlikely to be due to the physiological adaptation of the newborn rat to the postnatal environment. Cardiac deceleration or acceleration in response to stimulation, as demonstrated in the present study, traditionally has been related to central sensory processes, such as the orienting response and defensive response (27,38). Given the differences in stimuli and developmental gradation from bradycardia to tachycardia, the relationship of perinatal HR responses to organizing concepts of sensory processing remains uncertain, suggesting the need to employ multiple, independent measures of sensory responsiveness in fetuses and neonates. ACKNOWLEDGEMENTS This research is supported by NICH&HD (NIH) grant HD 16102 and Research Career DevelopmentAward HI:) 00719 to W.P.S.A.E.R is supported by NIMH grant MH 46485. J.R.A is supported by NIMH grant MH 28355. P.G.H is supported by grants from the Wellcome Trust, NuffleldFoundationand PhysiologicalSociety of Great Britain.

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